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Impact of serum storage conditions on microRNA stability

Leukemia volume 26, pages 2414–2416 (2012)Cite this article

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As microRNAs (miRNAs) were found to be present in human serum in a remarkably stable and cell-independent form, their potential as novel noninvasive biomarkers for physiological and pathophysiological conditions, including cancer, is of growing interest.1, 2 Serum miRNAs can be found in exosomes3 and bound to proteins, such as AGO2,4 increasing their stability and circulation time. However, owing to limited space and availability of −80 °C freezers, many hospitals and laboratories store patient serum samples at −20 °C, for many years. We therefore investigated the impact of short-term, intermediate-term and long-term storage at −80 and −20 °C on the stability and abundance of individual and total miRNAs in human serum.

Serum was obtained from coagulated whole blood after centrifugation at 2000, r.p.m. for 10 min. All patients gave informed consent for the research use of their specimens, in accordance with the Declaration of Helsinki. The miRNA fraction was quantified using the Bioanalyzer small RNA kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. This method has a detection range of 50–2000 pg/μl (www.genomics.agilent.com). All samples were analyzed in triplicates and on separate chips to identify chip-to-chip variations. In addition, the expression of selected miRNAs was tested by quantitative real time PCR (qRT-PCR) using Taqman amplicons (Life Technologies, Darmstadt, Germany). Four individual miRNAs (miR-223, miR-451, miR-93 and miR-24) were selected on the basis of their expression in hematopoietic cells and amplification efficacy.5 miR-223 and miR-451 are highly abundant in mature myeloid6 and red blood cells,5 respectively, whereas miR-24 is ubiquitously expressed in all hematopoietic lineages.5 miR-93 was included because of its previously published use as a housekeeping gene in serum samples.7 RNA was extracted from 200 μl of serum using Qiagen miRNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, because of its superior efficacy and handling compared with other common RNA extraction protocols (results not shown). cDNA synthesis as well as qRT-PCR was performed according to the manufacturer’s instructions and all samples were analyzed in triplicate. Mean absolute Ct values and s.d. are displayed on the graphs. Statistics were performed using Student’s t-test and P<0.05 was considered as significant.

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Acknowledgements

This study was supported by the Deutsche Krebshilfe grant (109420), the European Hematology Association fellowship (2010/04) and by the Deutsche Forschungsgemeinschaft grant (KU2288/3-1).

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Author notes

  1. S Grasedieck and N Schöler: These authors contributed equally to this work.

  2. C Langer and Florian Kuchenbauer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany

    S Grasedieck, M Bommer, A Rouhi, J Bloehdorn, D Mertens, H Döhner, C Langer & Florian Kuchenbauer

  2. Klinische Kooperationseinheit Dermato-Onkologie, German Cancer Research Center, Heidelberg, Germany

    N Schöler

  3. Department of Internal Medicine I, University Hospital of Ulm, Ulm, Germany

    J H Niess

  4. Department of Neurology, University Hospital of Ulm, Ulm, Germany

    H Tumani

  5. Oncological Clinic Moers, Moers, Germany

    P Liebisch

  6. Institute of Experimental Cancer Research, Comprehensive Cancer Centre, University Hospital of Ulm, Ulm, Germany

    C Buske & Florian Kuchenbauer

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  1. S Grasedieck
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  2. N Schöler
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  13. Florian Kuchenbauer
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Corresponding author

Correspondence to Florian Kuchenbauer.

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Grasedieck, S., Schöler, N., Bommer, M. et al. Impact of serum storage conditions on microRNA stability. Leukemia 26, 2414–2416 (2012). https://doi.org/10.1038/leu.2012.106

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